Table 1.
Details on immunofluorescence stainings on human cryosections.
| Procedure | MASP-2 | Agrin | Heparan sulfate | Activated C3 |
|---|---|---|---|---|
| Fixation | Icecold 100% aceton for 10 min | Icecold 100% aceton for 10 min | Icecold 100% aceton for 10 min | Icecold 100% aceton for 10 min |
| Peroxidase inactivation | 0.03% H2O2 in PBS for 30 min in the dark | – | – | – |
| Blocking a-specific background | 1% BSA in PBS for 15 min | 1% BSA in PBS for 15 min | 1% BSA in PBS for 15 min | 1% BSA in PBS for 15 min |
| Blocking endogenous biotin | – | – | Avidin/Biotin blocking kit (SP-2001; Vector Laboratories; Burlingame CA, USA) | – |
| Primary antibody | Rat mAb anti-human MASP-2, clone 8B5, 1:100 (Hycult, Uden, The Netherlands) | Mouse mAb JM-72 anti-human agrin, 1:750 (43) | Biotinylated mouse mAb anti-heparan sulfate, clone 10E4, 1:50 (Amsbio, Abingdon, UK) | Mouse IgG2a mouse mAb anti-neoepitope on C3b, iC3b and C3c, clone bH6, 1:200 (Hycult) |
| Secondary antibody | Rabbit anti-rat IgG-HRP, 1:200 + 5% normal human serum for (Dako, Glostrup, Denmark) | Donkey anti-Mouse IgG-Alexa 488, 1:250 (Life Technologies, Carlsbad CA, USA) | – | Donkey anti-mouse IgG-Alexa647, 1:250 (Life Technologies, Carlsbad CA, USA) |
| Tertiary antibody | Goat anti-rabbit IgG-HRP, 1:200 + 5% normal human serum (Dako) | – | – | – |
| Amplification HRP signal | TSA Tyramide-TRITC, 1:50 for 10 min (PerkinElmer, Waltham MA, USA) | – | – | – |
| Detection biotinylated antibody | – | – | Streptavidin-FITC, 1:300 (Invitrogen, Waltham MA, USA) | – |
| Nuclear staining | Dapi for 10 min | Dapi for 10 min | Dapi for 10 min | Dapi for 10 min |
| Embedment | Citifluor (Haffield PA, USA) | Citifluor (Haffield PA, USA) | Citifluor (Haffield PA, USA) | Citifluor (Haffield PA, USA) |
Stainings are performed as MASP2/agrin double staining, as MASP2/HS double staining, and as MASP2/HS/actC3 triple staining.
All antibody incubations were done for 30 min at room temperature in PBS + 1% BSA. Sections were washed with PBS in between the incubation steps. Double- and triple stainings were designed in such a way that all cross-reactions among the stainings were avoided.